Pseudomonas putida P683 and application thereof in saline-alkali soil improvement

Abstract

The invention discloses pseudomonas putida P683 and application thereof in saline-alkali soil improvement, and relates to the technical field of microorganisms, and the technical key points are as follows: the pseudomonas putida P683 is specifically pseudomonas putida P683 and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.27636, and the preservation date is June 15, 2023; the 16S rRNA (ribosomal Ribonucleic Acid) gene sequence of the pseudomonas P683 is as shown in Seq ID (Identity) No. 1. According to the novel acid-producing and alkali-reducing pseudomonas P683 and the fermentation liquor thereof, the strain P683 can produce various organic acids and reduce the pH value of the environment, so that the strain can grow in a high-alkali environment. The strain P683 fermentation liquor can effectively reduce the pH value of soda saline-alkali soil and relieve alkaline stress, the strain P683 and the fermentation liquor thereof not only can effectively improve saline-alkali soil and increase the emergence rate of crops, but also have safety, environmental protection and feasibility of industrial production, and a new and effective solution is provided for saline-alkali soil improvement.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Pseudomonas putrefaciens P683 and application thereof in improving saline-alkali soil. Background Art

[0002] Saline-alkali soil is a general term for saline soil and alkaline soil. According to the severity of salinization, it can usually be divided into mild, moderate and severe saline-alkali soil. Saline-alkali soil is a barrier soil that is widely distributed throughout the world. According to incomplete statistics, the area of ​​saline-alkali land in the world is 954.38 million hectares, accounting for about 10% of the total land area in the world. The area of ​​saline-alkali soil in my country is 36 million hectares, accounting for 4.9% of the developable land area. Saline-alkali land is widely distributed in my country, and more than 30% of saline-alkali land is distributed in the northeast, northwest and coastal areas. Saline-alkali soil seriously affects the root growth of plants, reduces the utilization rate of cultivated land, and has a negative impact on the agricultural economy. In addition, the salt in saline-alkali land will migrate with water, which may lead to salinization of groundwater and surface water, affect the quality of water resources, and pose a potential threat to human drinking water and irrigation water.

[0003] Soil is the foundation of agricultural production, and saline-alkali soil is one of the important reserve arable land resources in my country. In recent years, the total demand for grain in my country has continued to grow. The improvement and management of saline-alkali land has great practical significance for regional ecological restoration and reconstruction, local economic stability and development, and national food security. At present, saline-alkali soil improvement includes physical improvement, chemical improvement, and biological improvement. Among them, biological improvement has received widespread attention due to its effectiveness and sustainability. Salt-alkali stressed soils often exist in high pH environments, and lowering soil pH is the key to the reclamation of salinized soils. Screening and applying acid-producing microorganisms to lower the pH of saline-alkali soils has become an effective and sustainable biological means to improve saline-alkali soils.

[0004] Therefore, the present invention aims to provide a Pseudomonas putrefaciens P683 and application thereof in improving saline-alkali soil. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and provide a Pseudomonas odoriferus P683 and its application in saline-alkali soil improvement, aiming to solve the problem of saline-alkali soil improvement. Pseudomonas odoriferus P683 strain can produce a variety of organic acids, reduce the pH value of the environment, and enable it to grow in a high alkaline environment.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: a Pseudomonas tataricus P683, the strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit number is CGMCC No.27636, and the deposit date is June 15, 2023; the 16S rRNA gene sequence of the Pseudomonas P683 is shown in Seq ID No.1.

[0007] Another object of the present invention is to provide a fermentation broth of the above-mentioned Pseudomonas putrefaciens P683, wherein the fermentation broth is produced during the culture process of the Pseudomonas P683 strain and contains bacterial cells and substances secreted into the culture medium.

[0008] Another object of the present invention is to provide a method for preparing the fermentation broth of Pseudomonas rottenis P683, wherein the Pseudomonas rottenis P683 strain is inoculated into NB culture medium and cultured at 28°C and 220 rpm with shaking until saturation.

[0009] Another object of the present invention is to provide a method for lowering the pH value of the environment by using Pseudomonas odorifera P683 or its fermentation broth, wherein the Pseudomonas P683 and / or the fermentation broth of Pseudomonas P683 are used to produce a variety of organic acids, lower the pH value of the environment, and enable the strain to grow in a high alkaline environment.

[0010] Another object of the present invention is to provide a method for improving soda saline-alkali soil by using Pseudomonas putrefaciens P683 or its fermentation liquid, wherein the Pseudomonas P683 and / or the fermentation liquid of Pseudomonas P683 are used to effectively reduce the pH value of the soda saline-alkali soil and alleviate alkaline stress.

[0011] Another object of the present invention is to provide a method for improving the emergence rate of corn in saline-alkali soil by using Pseudomonas rotundus P683 or its fermentation liquid, wherein the Pseudomonas P683 and / or the fermentation liquid of Pseudomonas P683 are used for coating and treating corn seeds, thereby significantly improving the emergence rate of corn in saline-alkali soil.

[0012] Another object of the present invention is to provide a method for improving saline-alkali soil and increasing the emergence rate of crops in saline-alkali soil using Pseudomonas putrefaciens P683 or its fermentation liquid, and the application of Pseudomonas P683 and / or the fermentation liquid of Pseudomonas P683 in improving saline-alkali soil and increasing the emergence rate of crops in saline-alkali soil.

[0013] Compared with the prior art, the beneficial effects of this scheme are as follows: the present invention provides a Pseudomonas odoriferus P683 and its application in saline-alkali soil improvement, which is specifically aimed at the problem of saline-alkali soil improvement. The Pseudomonas odoriferus P683 strain can produce a variety of organic acids, effectively reduce the environmental pH value, and enable it to grow in a high-alkaline environment, which is of great significance for the agricultural utilization of saline-alkali land. First, the fermentation liquid of Pseudomonas odoriferus P683 can significantly reduce the pH value of soda saline-alkali soil from alkaline to nearly neutral, which is crucial for alleviating soil alkaline stress, improving soil fertility and crop growth conditions. Compared with traditional physical and chemical improvement methods, this biological method for improving saline-alkali soil has the advantages of low cost, good effect, and environmental friendliness. In addition, the emergence rate of seeds in saline-alkali soil can be significantly improved by coating corn seeds with fermentation liquid, which has a direct promoting effect on increasing crop yields and agricultural economic benefits.

[0014] Secondly, the application of Pseudomonas putrefaciens P683 not only improves the agricultural value of saline-alkali soil, but also has safety and environmental protection. The strain is harmless to humans and animals, does not cause environmental pollution, and meets the requirements of modern green agriculture and environmental protection. In addition, due to the simple culture conditions and easy preservation, the strain is suitable for industrial production, which helps to reduce costs and improve efficiency, making the technical solution of the present invention more easily adopted and applied. These characteristics make the present invention have a wide range of application prospects and significant ecological and economic benefits in the field of saline-alkali soil improvement.

[0015] In summary, the present invention provides a new type of Pseudomonas putrefaciens P683 and its fermentation liquid, which can not only effectively improve saline-alkali soil and increase crop emergence rate, but also has safety, environmental protection and feasibility of industrial production, providing a new and effective solution for saline-alkali soil improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a litmus plate test diagram of the acid production characteristics of Pseudomonas P683 in Example 2 of the present invention;

[0017] Figure 2 It is a growth curve and pH value monitoring diagram of the fermentation process of Pseudomonas P683 in Example 2 of the present invention;

[0018] Figure 3 It is the HPLC detection spectrum of organic acids in the concentrated supernatant of Pseudomonas P683 fermentation in Example 3 of the present invention;

[0019] Figure 4 is a graph showing the growth and pH value dynamic monitoring of Pseudomonas sp. P683 in a high alkaline medium in Example 4 of the present invention;

[0020] Figure 5This is a soil pH value detection diagram after the Pseudomonas P683 fermentation liquid treats saline-alkali soil in Example 5 of the present invention;

[0021] Figure 6 This is a diagram for detecting the corn emergence rate after the corn seeds were coated with the Pseudomonas P683 fermentation liquid in Example 6 of the present invention. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0023] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. In addition, the experimental methods in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources to repeat the experiments of this application unless otherwise specified.

[0024] The present invention will be described in detail below with reference to embodiments.

[0025] Example 1: Pseudomonas odorifera P683

[0026] A Pseudomonas taetrolens P683, deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC), with a deposit number of CGMCC No. 27636, a deposit date of June 15, 2023, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with a postal code of 100101; the Pseudomonas taetrolens P683 is an aerobic Gram-negative bacterium that can produce fluorescence on King's B medium.

[0027] The 16S rRNA gene sequence of Pseudomonas sp. strain P683 is shown in Seq ID No.1.

[0028] In this embodiment, a fermentation broth of Pseudomonas putrefaciens P683 is also provided: the fermentation broth is produced during the culture process of the Pseudomonas P683 strain and contains bacterial cells and substances secreted into the culture medium;

[0029] The preparation method of the fermentation liquid is as follows:

[0030] Pseudomonas P683 strain was inoculated into NB medium and cultured at 28°C and 220 rpm with shaking until saturation, which was the fermentation broth of strain P683.

[0031] The Pseudomonas P683 or / and the fermentation broth of Pseudomonas P683 provided in this embodiment can be used to produce a variety of organic acids, reduce the environmental pH value, and enable the strain to grow in a high-alkaline environment; effectively reduce the pH value of soda saline-alkali soil and alleviate alkaline stress; and coat corn seeds to significantly improve the emergence rate of corn in saline-alkali soil.

[0032] The following is the method for preparing the culture medium used in the examples, as follows:

[0033] Nutrient Agar (NA) medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, 12 g agar, pH 7.0, distilled water to 1000 mL.

[0034] Nutrient Broth (NB) medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, pH 7.0, distilled water to 1000 mL.

[0035] Kang's B (KB) solid culture medium: 20 g of peptone No. 3, 0.4 g of MgSO4·7H2O, 10 mL of glycerol, 1.5 g of K2HPO4, 12 g of agar, and distilled water to make up to 1000 mL.

[0036] Kang's B (KB) liquid culture medium: 20 g of peptone No. 3, 0.4 g of MgSO4·7H2O, 10 mL of glycerol, 1.5 g of K2HPO4, and distilled water to make up to 1000 mL.

[0037] Example 2: Detection of acid production characteristics of Pseudomonas taeniasis P683

[0038] Litmus solution was used to detect the acid production of strain P683. Litmus solution, as an acid-base indicator, turns red under acidic conditions and turns blue under alkaline conditions. 0.1% litmus solution was added to NA solid culture medium to prepare a plate. Strain P683 was cultured in KB liquid culture medium overnight with shaking until saturation. 10 μL of the bacterial solution was spotted in the center of the plate. After drying, it was placed in a 28°C incubator and the color changes around the colonies were observed. At the same time, strain P683 was streaked on the plate and the color changes around the colonies were also observed. The results showed that a red halo appeared around the colonies of strain P683, as shown in the attached Figure 1 As shown, it indicates that strain P683 produces and secretes acidic substances during the culture process, making the surrounding culture medium acidic, and litmus solution turns red under acidic conditions.

[0039] In order to further detect the changes in acid production of strain P683 during its growth, strain P683 was inoculated into a conical flask containing 50 mL of NB medium, with an initial inoculation concentration of OD 600 =0.05, placed in a shaker at 28°C and 220 rpm, and the absorbance of the fermentation liquid at 600 nm (OD 600 The results showed that strain P683 entered the stable phase after 12 h of culture, and the pH dropped from neutral to below 4 and reached stability. Figure 2 As shown, it indicates that strain P683 indeed produced acidic substances during the fermentation process, which reduced the pH value of the environment.

[0040] Example 3: Identification of organic acids produced by Pseudomonas taeniasis P683

[0041] In order to identify the acidic substances produced and secreted by strain P683 during the fermentation process, the fermentation supernatant of strain P683 was freeze-dried and concentrated and then tested by high performance liquid chromatography (HPLC). The specific method is: strain P683 was inoculated into 50mL NB medium, and the initial inoculation volume was OD 600 =0.01, placed in a shaker at 28°C and 220rpm for 24h. The fermentation supernatant was collected by centrifugation, filtered through a 0.22μm filter membrane, and 24mL of the supernatant was vacuum freeze-dried. The lyophilized powder was re-dissolved in 1mL of sterile water and filtered through a 0.22μm filter membrane to obtain the fermentation supernatant concentrate (concentrated 24 times). The 11 organic acids in the fermentation supernatant concentrate were quantitatively detected using a high performance liquid chromatograph (Thermo Fisher U3000) to obtain the organic acid content information. The results showed that strain P683 produced a variety of organic acids during the fermentation process, such as Figure 3 As shown in Table 1, gluconic acid was produced the most, with the concentration reaching 1.16 mg / mL before concentration. In addition, strain P683 also produced more lactic acid and acetic acid, with the concentrations reaching 0.60 mg / mL and 0.32 mg / mL respectively before concentration, as shown in Table 1.

[0042] Table 1 HPLC detection of organic acid quantitative results

[0043]

[0044] Note: “ND” means not detected

[0045] Example 4: Growth of Pseudomonas taeniae P683 in high alkaline medium and dynamic monitoring of pH value

[0046] In order to test whether strain P683 can grow in an alkaline environment and effectively reduce the alkaline pH value, strain P683 was inoculated into 50 mL NB medium (pH = 10) with an initial inoculum size of OD 600 =0.05, placed in a shaker at 28°C, 220 rpm, and cultured. OD was measured every 12 h. 600 The results showed that in the alkaline NB medium with pH = 10, strain P683 was in the delayed period for the first 12 hours and basically did not grow; from 12 hours to 24 hours, it was in the logarithmic growth phase and began to grow rapidly; and it reached the stable period at 48 hours. The pH value dropped rapidly to below 4 from 12 hours to 36 hours, and was stable after 36 hours. Figure 4 The above results show that strain P683 can grow in a high alkaline environment and reduce the alkaline pH value by producing acid, revealing its application potential of acid production and alkalinity reduction.

[0047] Example 5: Effect of Pseudomonas putrefaciens P683 on pH value of soda saline-alkali soil

[0048] In order to test the acid-reducing and alkaline-reducing properties of strain P683 and its application in saline-alkali soil improvement, the fermentation liquid (OD 600 =0.231pH=3.92) was mixed with soda saline-alkali soil (pH=10.22) in the Northeast region in a ratio of 1:1, left to stand at room temperature for 1 day, and the pH value of the treated soil was tested in accordance with the National Environmental Protection Standard of the People's Republic of China (HJ 962-2018). The results showed that at 0 days after treatment, the pH value of the soil in the group treated with the fermentation liquid of strain P683 dropped to 7.96, which was significantly lower than the pH value of the soil in the group treated with H2O (pH=10.14). After standing at room temperature for 1 day, the pH value of the soil in the group treated with the fermentation liquid of strain P683 further dropped to 6.75, becoming neutral. Figure 5 The results show that the fermentation broth of strain P683 has application potential in improving saline-alkali soil, especially in reducing the pH value of saline-alkali soil.

[0049] Example 6: Effect of Pseudomonas rotundus P683 coating on corn seeds on their germination rate in soda saline-alkali soil

[0050] In this example, corn was used as a model crop to test the effect of coating corn seeds with fermentation liquid of strain P683 on their emergence rate in saline-alkali soil. Some colonies of strain P683 were selected and inoculated into 5 mL of NB medium, and then placed in a shaking incubator at 28°C and 220 rpm for 12 h to obtain the fermentation liquid of strain P683 (OD 600=0.877pH=3.49). The fermentation liquid of strain P683 was used to coat corn seeds, and sterile water was used as a blank control. The specific coating method is: add a film-forming agent with a final concentration of 8% to the bacterial liquid P683 and sterile water and mix them evenly to obtain the coating agent; pick an appropriate number of complete and full corn seeds and weigh them; mix the prepared coating agent according to the mass ratio of coating agent to seeds of 1:30, stir well, and dry in the shade to obtain coated corn seeds. The coated corn seeds were planted in pots filled with soda saline-alkali soil (pH=10.22), 16 seeds / pot, and 3 pots were planted for each treatment as a replicate. They were placed in a solar greenhouse for cultivation, and the emergence of seedlings was observed and counted. On the 18th day after planting, the emergence rate of corn under the two treatments was statistically analyzed. The results showed that compared with the H2O coating treatment, the emergence rate of corn seeds coated with the fermentation liquid of strain P683 was significantly improved. Figure 6 shown.

[0051] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A Pseudomonas odorifera P683, characterized in that: The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.27636 and a deposit date of June 15, 2023; the 16S rRNA gene sequence of the Pseudomonas P683 is shown in Seq ID No.

1.

2. A fermentation broth of Pseudomonas taeniae P683 as claimed in claim 1, characterized in that: The fermentation broth is produced by the Pseudomonas P683 strain during the culture process and contains bacterial cells and substances secreted into the culture medium.

3. A method for preparing the fermentation broth of Pseudomonas taeniae P683 as claimed in claim 2, characterized in that: Pseudomonas P683 strain was inoculated into NB medium and cultured at 28°C and 220 rpm with shaking until saturation.

4. A method for reducing the pH value of an environment by using Pseudomonas rotundus P683 or its fermentation liquid, characterized in that: The Pseudomonas P683 or / and the fermentation liquid of Pseudomonas P683 are used to produce a variety of organic acids, reduce the pH value of the environment, and enable the strain to grow in a high-alkaline environment.

5. A method for improving soda saline-alkali soil by using Pseudomonas putrefaciens P683 or its fermentation liquid, characterized in that: The Pseudomonas P683 and / or the fermentation liquid of Pseudomonas P683 are used to effectively reduce the pH value of the soda saline-alkali soil and alleviate alkaline stress.

6. A method for improving the emergence rate of corn in saline-alkali soil by using Pseudomonas rotundus P683 or its fermentation liquid, characterized in that: The Pseudomonas P683 or / and the fermentation liquid of Pseudomonas P683 are used for coating and treating corn seeds, thereby significantly improving the emergence rate of corn in saline-alkali soil.

7. A method for improving saline-alkali soil and increasing the emergence rate of crops in saline-alkali soil by using Pseudomonas rotundus P683 or its fermentation liquid, characterized in that: The Pseudomonas P683 or / and the fermentation liquid of Pseudomonas P683 are used in improving saline-alkali soil and improving the emergence rate of crops in saline-alkali soil.

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